CN108660173A - A kind of method that ultrasonic wave added peony seeds cake protein enzymolysis prepares anti-oxidation peptide - Google Patents
A kind of method that ultrasonic wave added peony seeds cake protein enzymolysis prepares anti-oxidation peptide Download PDFInfo
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- CN108660173A CN108660173A CN201810492178.0A CN201810492178A CN108660173A CN 108660173 A CN108660173 A CN 108660173A CN 201810492178 A CN201810492178 A CN 201810492178A CN 108660173 A CN108660173 A CN 108660173A
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- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a kind of methods that ultrasonic wave added peony seeds cake protein enzymolysis prepares anti-oxidation peptide, by being handled using pulse ultrasonic wave enzymatic hydrolysis system after enzymolysis starts, with by same enzymolysis process but compared with not applying the check experiment of ultrasound, the peptide yield of enzymolysis product can be made to improve 12%~25%, removing the EC50 values of DPPH reduces by 15%~38%, substantially increases the activity of enzymolysis efficiency and product.
Description
Technical field
The present invention relates to biotechnologies, and in particular to a kind of ultrasonic wave added peony seeds cake protein enzymolysis prepares anti-oxidant
The method of peptide.
Background technology
Fat content is 29~34% in peony seeds, the relative amount point of the linolenic and linoleic beneficial to health
It Wei 57.9% and 28.1%.Ministry of Public Health's approval peony seed oil in 2011 is as new resource food, hereafter the oil plantation of tree peony
It is had received widespread attention with utilizing.Currently, oil is about 300,000 mu or so with the cultivated area of tree peony, and show a rising trend.
According to the 300 kilograms of calculating of per mu yield peony seeds, 90,000 tons of peony seeds can be generated, it is annual to generate fats and oils processing waste --- tree peony
The seedcake dregs of rice are up to as many as 50,000 tons, are handled as waste and cause great pressure to environment, it is often more important that wherein had
The ingredient of better nutritivity and healthcare function is not developed, and causes the significant wastage of resource.Contain in peony seeds grouts
35% or so protein, amino acid composition is reasonable, is a kind of ideal, potential high-quality protein resource.If using its as
The raw material of active peptide is prepared, a kind of natural active peptide product can be not only developed, peony seeds processing industry chain can also be extended,
Greatly improve economic benefit.
Currently, people are successfully with casein, aquatic products, corn and soybean, egg, dairy products etc. for raw material, by egg
White enzyme Controlled-enzymatic Hydrolysis prepares active peptide, but above-mentioned raw materials are the life food of the mankind, and being used to prepare active peptide will certainly make
At the waste of grain.Active peptide, which is prepared, by carrying out enzymolysis to the albumen in peony seeds grouts can not only improve the attached of peony seeds
It is value added, and can solve the problems, such as that protein resource wastes.The method that the preparation of active peptide mainly uses protease hydrolytic, still
Conventional enzymolysis there are hydrolysis efficiencies it is low, hydrolysis rate is slow, enzyme utilization rate is low, production cost is high the shortcomings of.Utilize some physics hands
Section carries out pretreatment to substrate or the other materials for participating in reacting and tends to accelerate hydrolysis process.In recent years, ultrasonic technology
It is more and more extensive in the application of food processing field, using more especially in terms of the extraction of active ingredient, and utilize ultrasonic wave
The report that technology accelerates protein digestion is less.So far, there has been no ultrasonic wave auxiliary peony seeds cake protein enzymolysis to prepare work
The report of property peptide.
Invention content
The technical scheme is that：
A kind of method that ultrasonic wave added peony seeds cake protein enzymolysis prepares anti-oxidation peptide, which is characterized in that including walking as follows
Suddenly：
(1) the peony seeds dregs of rice are crushed, is 1 according to the mass volume ratio of peony seeds grouts powder and water:5~1:The ratio of 20g/mL
Example is mixed；
(2) protease for accounting for raw material peony seeds grouts quality 0.5%~6% is added, passes through dropwise addition 0.5mol/LNaOH
Solution adjusts pH value to 7.0, adjusts the temperature to 50 DEG C, and 10 are handled using impulse ultrasound is carried out to enzymatic hydrolysis system after enzymolysis starts
~60min, treatment conditions are：Frequency 20kHz, power 10W/L~50W/L, work/intermittently are than for 0.5s/1s~5s/1s；It is super
After sonication stops, continuing to digest, digests 1~3 hour total time；
(3) after digesting, enzymolysis liquid is heated to 90 DEG C and keeps 5min to enzyme deactivation, enzymolysis liquid is then cooled to room
Temperature takes supernatant to carry out being concentrated in vacuo to dry substance concentration after adjusting material liquid pH value to 7.0,4500rpm centrifugations, 10~20min
For 30%w/w, concentrate is freeze-dried or is spray-dried obtained peony seeds proteolysis Gly-His-Lys.
Further, the protease described in step (2) is neutral proteinase, trypsase, compound protease, alkaline egg
White enzyme.
Beneficial effects of the present invention：
The present invention is handled enzymatic hydrolysis system using pulse ultrasonic wave after enzymolysis starts, with the same enzymolysis process of process but not
The check experiment for applying ultrasound is compared, and the peptide yield of enzymolysis product can be made to improve 15%~25%, remove the EC50 values of DPPH
15%~38% is reduced, the activity of enzymolysis efficiency and product is substantially increased.
Specific implementation mode
Below by non-limiting embodiment, the present invention is further explained, understands the present invention.
Anti-oxidant (activity) peptide can remove free radical, suppress or eliminate and slow down oxidation reaction.Its antioxidation mechanism
Including：Hydrogen, buffered physiologic pH value, chelated metal ions and capture free radical etc. are provided to antioxidase.Pass through survey in the present invention
Determine the antioxidant activity that enzymolysis product evaluates the Scavenging activity of DPPH free radicals peony seeds cake protein enzymolysis product.The peony seeds dregs of rice
Proteolysis peptide product is configured to gradient concentration solution, measures the clearance rate to DPPH free radicals, and then calculate EC50 values.
The assay method of DPPH free radical scavenging activities：Peony seeds proteolysis peptide is configured to the solution of various concentration, is taken
The DPPH ethanol solutions of a concentration of 0.04g/L of 2mL are added in each 2mL of various concentration solution, are uniformly mixed, and react 20min,
4000rpm centrifuges 20min, and supernatant is taken to measure light absorption value Ai at 517nm；Above-mentioned each strength solution 2mL is taken respectively, is added
2mL absolute ethyl alcohols measure light absorption value Aj after reaction；It is reacted with the DPPH solution of a concentration of 0.04g/L of 2mL and 2mL absolute ethyl alcohols
Light absorption value A0 is measured as reference.Clearance rate calculation formula is：
EDPPH=[1- (Ai-Aj)/A0] × 100
In formula：EDPPH is the clearance rate to DPPH free radicals, %；Ai is the DPPH solution that 2mL is added in 2mL sample solutions
Light absorption value；Aj is the light absorption value that 2mL absolute ethyl alcohols are added in 2mL sample solutions；It is anhydrous that 2mL is added in the DPPH solution that A0 is 2mL
The light absorption value of ethyl alcohol.
The calculating of EC50：The concentration that EC50 is peony seeds proteolysis peptide when reaching 50% to DPPH clearance rates.First
Sample is diluted to a series of concentration, measures clearance rate of each concentration samples to DPPH, draws the relation curve of clearance rate and concentration,
The EC50 values of sample are found on curve.
Experiment is shown in Table 1 with the action condition manufacturer of 4 kinds of commercial proteases.
The action condition of 1 experiment protease of table and manufacturer
With reference to specific embodiment, the present invention is further elaborated.
Embodiment 1
By the peony seeds grouts after crushing with water 1:The ratio of 5g/mL mixes, and addition accounts for peony seeds grouts quality 0.5%
Neutral proteinase digested, hydrolysis temperature be 60 DEG C, pH 7.0, after enzymolysis starts to enzymatic hydrolysis system use frequency
20kHz, power 10W/L, work/intermittently stop being ultrasonically treated, continue enzyme than handling 10min for the impulse ultrasound of 0.5s/1s
Solution digests 1 hour total time.After enzymolysis, enzymolysis liquid is heated to 90 DEG C of holding 5min enzyme deactivations, is cooled to room temperature,
Supernatant is carried out being concentrated in vacuo to dry substance concentration being 30% (w/w), obtained by freeze drying after 4500rpm centrifugations 15min
Peony seeds proteolysis peptide product measures the yield of polypeptide products and to DPPH using the assay method of DPPH free radical scavenging activities
Clearance rate.
Check experiment 1
Operating procedure is same as Example 1, but does not apply ultrasonication after enzymolysis starts, and measures obtaining for polypeptide products
Rate and clearance rate to DPPH.
Embodiment 2
By the peony seeds grouts after crushing with water with mass volume ratio 1:The ratio of 20g/mL is mixed, to account for raw material
The ratio of peony seeds grouts weight 6% is added neutral proteinase and is digested, and hydrolysis temperature is 60 DEG C, and pH 7.0 is opened in enzymolysis
Frequency 20kHz used to enzymatic hydrolysis system after beginning, power 50W/L, work/intermittently than handling 60min for the impulse ultrasound of 5s/1s,
Stop being ultrasonically treated, continue to digest, digests 3 hours total times.After enzymolysis, enzymolysis liquid is heated to 90 DEG C, 5min is kept to go out
Enzyme is cooled to room temperature, and carries out being concentrated in vacuo to dry substance concentration being 30% (w/w) by supernatant after 4500rpm centrifugations 10min,
Spray-dried obtained peony seeds proteolysis peptide product, measures the yield of polypeptide products and the clearance rate to DPPH.
Check experiment 2
Operating procedure is same as Example 2, but does not apply ultrasonication after enzymolysis starts, measure polypeptide products
Yield and clearance rate to DPPH.
Embodiment 3
By the peony seeds grouts after crushing with water with mass volume ratio 1:The ratio of 13g/mL is mixed, to account for raw material
The ratio of peony seeds grouts weight 3% is added trypsase and is digested, and hydrolysis temperature is 50 DEG C, and pH 8.0 starts in enzymolysis
Frequency 20kHz used to enzymatic hydrolysis system afterwards, power 35W/L, work/intermittently than handling 35min for the impulse ultrasound of 3s/1s, stop
It is only ultrasonically treated, continues to digest, digest 2 hours total times.After enzymolysis, enzymolysis liquid is heated to 90 DEG C, 5min is kept to go out
Enzyme is cooled to room temperature, and is adjusted and is carried out supernatant after material liquid pH centrifuges 10min to 7.0,4500rpm to be concentrated in vacuo to dry matter
A concentration of 30% (w/w), spray-dried obtained peony seeds proteolysis peptide product, measures the yield of polypeptide products and to DPPH
Clearance rate.
Check experiment 3
Operating procedure is same as Example 3, but does not apply ultrasonication after enzymolysis starts, and measures obtaining for polypeptide products
Rate and clearance rate to DPPH.
Embodiment 4
By the peony seeds grouts after crushing with water with mass volume ratio 1:The ratio of 15g/mL is mixed, to account for raw material
The ratio of peony seeds grouts weight 3% is added compound protease and is digested, and hydrolysis temperature is 50 DEG C, and pH 6.5 is opened in enzymolysis
Frequency 20kHz used to enzymatic hydrolysis system after beginning, power 30W/L, work/intermittently than handling 20min for the impulse ultrasound of 2s/1s,
Stop being ultrasonically treated, continue to digest, digests 1.5 hours total times.After enzymolysis, enzymolysis liquid is heated to 90 DEG C of holding 5min
Enzyme deactivation is cooled to room temperature, and is adjusted and is carried out supernatant after material liquid pH centrifuges 15min to 7.0,4500rpm to be concentrated in vacuo to dry
Matter a concentration of 30% (w/w), spray-dried obtained peony seeds proteolysis peptide product measure the yield of polypeptide products and right
The clearance rate of DPPH.
Check experiment 4
Operating procedure is same as Example 4, but does not apply ultrasonication after enzymolysis starts, and measures obtaining for polypeptide products
Rate and clearance rate to DPPH.
Embodiment 5
By the peony seeds grouts after crushing with water with mass volume ratio 1:The ratio of 10g/mL is mixed, to account for raw material
The ratio of peony seeds grouts weight 2% is added alkali protease and is digested, and hydrolysis temperature is 50 DEG C, and pH 9.0 is opened in enzymolysis
Frequency 20kHz used to enzymatic hydrolysis system after beginning, power 20W/L, work/intermittently than handling 40min for the impulse ultrasound of 3s/1s,
Stop being ultrasonically treated, continue to digest, digests 2 hours total times.After enzymolysis, enzymolysis liquid is heated to 90 DEG C, 5min is kept to go out
Enzyme is cooled to room temperature, and is adjusted and is carried out supernatant after material liquid pH centrifuges 20min to 7.0,4500rpm to be concentrated in vacuo to dry matter
A concentration of 30% (w/w), spray-dried obtained peony seeds proteolysis peptide product, measures the yield of polypeptide products and to DPPH
Clearance rate.
Check experiment 5
Operating procedure is same as Example 5, but does not apply ultrasonication after enzymolysis starts, and measures obtaining for polypeptide products
Rate and clearance rate to DPPH.
2 embodiment of table is compared with the polypeptide yield of check experiment and the EC50 values of removing DPPH free radicals
As can be seen from Table 2, enzymatic hydrolysis system is handled after enzymolysis starts using pulse ultrasonic wave, enzymolysis product can be made
Peptide yield improve 12%~25%, remove DPPH EC50 values reduce by 15%~38%, substantially increase enzymolysis efficiency and production
The activity of object.
Claims (2)
1. a kind of method that ultrasonic wave added peony seeds cake protein enzymolysis prepares anti-oxidation peptide, which is characterized in that include the following steps：
(1) the peony seeds dregs of rice are crushed, is 1 according to the mass volume ratio of peony seeds grouts powder and water:5~1:The ratio of 20g/mL into
Row mixing；
(2) protease for accounting for raw material peony seeds grouts quality 0.5%~6% is added, it is molten with the NaOH of 0.5mol/L by dropwise addition
Liquid adjust pH value to 7.0, adjust the temperature to 50 DEG C, after enzymolysis starts to enzymatic hydrolysis system using carry out impulse ultrasound processing 10~
60min, treatment conditions are：Frequency 20kHz, power 10W/L~50W/L, work/intermittently is than for 0.5s/1s~5s/1s；Ultrasound
After processing stops, continuing to digest, digests 1~3 hour total time；
(3) after digesting, enzymolysis liquid is heated to 90 DEG C and keeps 5min to enzyme deactivation, enzymolysis liquid is then cooled to room
Temperature takes supernatant to carry out being concentrated in vacuo to dry substance concentration after adjusting material liquid pH value to 7.0,4500rpm centrifugations, 10~20min
For 30%w/w, concentrate is freeze-dried or is spray-dried obtained peony seeds proteolysis Gly-His-Lys.
2. a kind of method that ultrasonic wave added peony seeds cake protein enzymolysis prepares anti-oxidation peptide according to claim 1, special
Sign is that the protease described in step (2) is neutral proteinase, trypsase, compound protease, alkali protease.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706208A (en) * | 2019-01-24 | 2019-05-03 | 山东贝世康生物科技有限公司 | A kind of method that water enzymatic isolation method extracts peony seed oil and tree peony protein peptides |
| CN110079573A (en) * | 2019-04-08 | 2019-08-02 | 江苏大学 | A kind of method that watermelon seeds albumen prepares anti-oxidant, hypoglycemic, antibacterial more active peptides |
| CN110117632A (en) * | 2019-04-08 | 2019-08-13 | 江苏大学 | A kind of method that ultrasonic in combination double enzymolysis improves watermelon seeds polypeptide antioxidative stabilizer |
| CN110710592A (en) * | 2019-11-16 | 2020-01-21 | 四川农业大学 | Method for improving antioxidant activity of walnut cake protein |
| CN113999885A (en) * | 2021-12-02 | 2022-02-01 | 河南科技大学 | Preparation method of peony stamen protein powder |
| CN114058662A (en) * | 2021-08-25 | 2022-02-18 | 华南农业大学 | Preparation method of endothelium corneum gigeriae galli enzymolysis polypeptide with antioxidation effect |
| CN114317659A (en) * | 2022-01-25 | 2022-04-12 | 杭州佳嘉乐生物技术有限公司 | Preparation method of soybean oligomeric active peptide powder with multiple biological activities |
| CN116478261A (en) * | 2023-04-27 | 2023-07-25 | 山东牡丹爱生物科技有限公司 | Anti-fatigue antioxidant peony peptide and preparation method thereof |
| CN116970673A (en) * | 2023-09-11 | 2023-10-31 | 山东大树达孚特膳食品有限公司 | Anti-fatigue peony peptide and preparation method thereof |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706208A (en) * | 2019-01-24 | 2019-05-03 | 山东贝世康生物科技有限公司 | A kind of method that water enzymatic isolation method extracts peony seed oil and tree peony protein peptides |
| CN109706208B (en) * | 2019-01-24 | 2021-04-02 | 山东贝世康生物科技有限公司 | Method for extracting peony seed oil and peony protein peptide by hydrolysis method |
| CN110079573A (en) * | 2019-04-08 | 2019-08-02 | 江苏大学 | A kind of method that watermelon seeds albumen prepares anti-oxidant, hypoglycemic, antibacterial more active peptides |
| CN110117632A (en) * | 2019-04-08 | 2019-08-13 | 江苏大学 | A kind of method that ultrasonic in combination double enzymolysis improves watermelon seeds polypeptide antioxidative stabilizer |
| CN110117632B (en) * | 2019-04-08 | 2022-07-22 | 江苏大学 | Method for improving antioxidant stability of watermelon seed polypeptide by combining ultrasonic and double enzymolysis |
| CN110710592A (en) * | 2019-11-16 | 2020-01-21 | 四川农业大学 | Method for improving antioxidant activity of walnut cake protein |
| CN114058662A (en) * | 2021-08-25 | 2022-02-18 | 华南农业大学 | Preparation method of endothelium corneum gigeriae galli enzymolysis polypeptide with antioxidation effect |
| CN113999885A (en) * | 2021-12-02 | 2022-02-01 | 河南科技大学 | Preparation method of peony stamen protein powder |
| CN114317659A (en) * | 2022-01-25 | 2022-04-12 | 杭州佳嘉乐生物技术有限公司 | Preparation method of soybean oligomeric active peptide powder with multiple biological activities |
| CN114317659B (en) * | 2022-01-25 | 2024-01-26 | 杭州佳嘉乐生物技术有限公司 | Preparation method of soybean oligopeptide powder with multiple biological activities |
| CN116478261A (en) * | 2023-04-27 | 2023-07-25 | 山东牡丹爱生物科技有限公司 | Anti-fatigue antioxidant peony peptide and preparation method thereof |
| CN116478261B (en) * | 2023-04-27 | 2023-10-27 | 德州蓝力生物技术有限公司 | Anti-fatigue antioxidant peony peptide and preparation method thereof |
| CN116970673A (en) * | 2023-09-11 | 2023-10-31 | 山东大树达孚特膳食品有限公司 | Anti-fatigue peony peptide and preparation method thereof |
| CN116970673B (en) * | 2023-09-11 | 2024-02-23 | 山东大树达孚特膳食品有限公司 | Anti-fatigue peony peptide and preparation method thereof |
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Application publication date: 20181016 |